1. Field of the Invention
The present invention relates to a recombinant protein for siRNA delivery, which allows the efficient intracellular and in vivo delivery of siRNA. More particularly, the present invention relates to a recombinant protein comprising (1) a capsid protein of HBV (Hepatitis B virus) and (2) a siRNA binding protein that is located in the interior cavity of the recombinant protein. In the recombinant protein, siRNAs of interest bind to the siRNA binding protein to be encapsulated within the recombinant protein particle, thereby providing stability against the external attack such as nucleases and achieving the efficient intracellular and in vivo delivery of siRNA by its release into the cytosolic space after cell uptake. Also, RGD peptides are introduced on the exterior surface of the recombinant protein, thereby effectively targeting cancer cells and tissues and achieving silencing of the target gene.
2. Description of the Related Art
RNAi (RNA interference) refers to a phenomenon of selectively inducing degradation of targeted mRNAs or suppressing target gene expression by intracellular introduction of a double stranded RNA consisting of a sense RNA homologous to mRNA of a target gene and an antisense RNA having a sequence complementary to the sense RNA. First discovered in nematodes, RNAi is a highly conserved biological process among animals and plants, such as yeasts, insects, plants and humans.
An RNAi-inducing entity, siRNA (small interference RNA) is a short, double-stranded RNA consisting of approximately 20 to nucleotides. Intracellular delivery of siRNA can suppress expression of a targeted mRNA having complementary base sequence to the siRNA. Thus, siRNA has been regarded as a revolutionary tool for manipulating target biological processes owing to its therapeutic effects on diseases, easy preparation and high target selectivity.
Currently, cancer, viral infections, autoimmune diseases and neurodegenerative diseases have been explored as promising disease targets of siRNAs. Clinical studies have demonstrated the potential of siRNAs as a therapeutic agent for age-related macular degeneration (bevasiranib; Opko Health, Inc., Miami, Fla., USA; phase III), and respiratory syncytial virus infection (ALN-RSV01; Alnylam, Cambridge, Mass., USA; phase II) (Melnikova I. Nat Rev Drug Discov 2007, 6, 863-864). Moreover, human cancer therapy via systemic delivery of siRNA using transferrin-tagged, cyclodextrin-based polymeric nanoparticles (CALAA-01; Calando Pharmaceuticals, Pasadena, Calif., USA; phase I) has been recently announced (Oh Y K. et al., Adv Drug Deliver Rev 2009, 61, 850-862).
However, siRNA has low stability and is quickly degraded in vivo, and the anionic nature of siRNA hinders it from permeating a cell membrane with negative charge, leading to low levels of siRNA transfer into intracellular compartments. Accordingly, there is a need to develop a technology for the preparation of an effective delivery system that facilitates intracellular transfer of siRNA. Thus, novel delivery systems, which enable prolonged circulation of siRNA with resistance against enzymatic degradation, high accessibility to target cells via clinically feasible administration routes, and optimized cytosolic release of siRNA after efficient cellular uptake, are indispensably required for the efficient intracellular delivery of siRNAs.
As a siRNA delivery system, a siRNA-expressing recombinant plasmid or viral vector has been generally used. Alternatively, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticle, polycation, or liposome-based systems have been mainly used. However, the viral delivery system is problematic in that it is limited by the size of gene to be introduced, and generates side effects induced by immunogenic surface proteins of the viral vector to cause safety problems. There are also drawbacks in that the delivery systems using such cationic molecules or synthetic polymers have lower transfection efficiency and might induce cytotoxicity during intracellular gene delivery.
Therefore, the present inventors have made an effort to develop a novel siRNA delivery carrier showing enhanced stability and cellular uptake by improving the problems of the known siRNA delivery carriers. As a result, the present inventors designed a recombinant protein delivery carrier, in which siRNA is encapsulated within a spherical virus capsid protein to be protected from the external nucleases, leading to the enhanced stability, and siRNA can be delivered into the cytosol of the target cells by facilitating endosomal escape. Further, RGD peptides, which specifically bind with cancer cells to facilitate cellular uptake, are introduced on the exterior surface of the recombinant protein delivery carrier, thereby providing cancer targeting capability. Furthermore, the present inventors demonstrated the excellent potential of the recombinant protein as a delivery carrier via tests on its siRNA affinity, cellular uptake efficiency, in vivo stability and cancer targeting capability, thereby completing the present invention.